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. 2021 Jan 12;12:301. doi: 10.1038/s41467-020-20540-2

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — A multiplexed single-cell secretion assay was used to measure cytokine/chemokine production in individual BMDMs stimulated for 48 h with media alone, 10 ng/ml LPS + 10 ng/ml IFN-γ, 100 ng/ml IL-4, or both. a Density scatter plots of single-cell secretion intensity (a.f.u.) across individual cells for Chi3l3 vs IL-6 (top) and Chi3l3 vs IL-12p40 (bottom). b Quantification of single- and double-positive cells after co-stimulation for Chi3l3 and IL-12p40 (left) and Chi3l3 and IL-6 (right). c Pairwise odds ratios for all secreted proteins measured in co-stimulated cells. Data are presented as the log₂(odds ratio). The color bar indicates the direction and magnitude of the association. Odds ratios with an associated p-value > 0.05 (determined by two-sided Fisher’s exact test) are set to 0. d-e UMAP visualization of single-cell secretion data for 5 proteins (Chi3l3, CCL5, IL-6, IL-12p40, and TNFα), colored by stimulation (d) or consensus cluster (e). f Quantification of the fraction of cells present in each consensus cluster identified by ensemble clustering. g Heatmap of secretion profiles for the five secreted proteins in d grouped by consensus cluster. Inset is a magnified version of the smaller clusters. All single-cell secretion data presented is pooled from two biological replicates. L + I asc. LPS + IFN-γ-associated; IL-4 asc. IL-4-associated. Source data are provided as a Source Data file.